Dressing

ABSTRACT

A wound dressing for release of one or more therapeutic ingredients wherein the therapeutic ingredients are contained in liposomes, said liposomes may comprising releasing means being triggered by a wound constituent and thereby releasing the therapeutic ingredients of the liposomes. The dressing further comprises wound exudates handling means and is suitable for moist wound healing.

form of a hydrogel. The absorbent material may preferably be in the formof a layer.

The absorbent layer may have an absorption capacity of 0.9% NaCl aqueoussolution at 37° C. of at least 0.05 g/cm², more preferred at least 0.1g/cm², and most preferred at least 0.2 g/cm², even most preferred atleast 0.4 g/cm². In one embodiment of the invention the absorption is atleast 0.6 g/cm².

The liposomes used in the present invention may be microscopic sphericalvesicles based on small vehicles of lipid bilayers with aqueousenvironment between the bilayers. Dependent on the constituents used andthe production method the liposomes can be made in a wide range in sizesand be either unilamellar (ULV), oligolamellar (OLV) or multilamellar(MLV).

Liposomes have been extensively investigated as drug carriers for skindelivery and parenteral delivery for many drugs. The liposomes form avery stable membrane thus providing a good protection of the activesubstance and thereby an increased flexibility of the design of thedressing may be achieved. An increased stability of the activeingredient, during production, sterilization and storage may beobtained. Furthermore, liposomes provide an opportunity to incorporateboth lipophilic and hydrophilic components without use of any additionalagents.

The liposomes may either disrupt slowly without any external mediator orcomprise releasing means being triggered by a wound constituent andthereby releasing the therapeutic ingredients of the liposomes.

Lipid components can be cholesterol or various phopholipids, lichitine.g. phosphatidyl choline, phosphatidyl ethanolamin, phosphatidyl serinand phosphatidyl inositol or mixtures thereof. As lipids may besubjected to oxidation, antioxidants may be added, e.g. tocopherol,butylhydroxitoluen, butylhydroxyianisol, ascorbic acid esters orsodiummetabisulfit.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to wound dressings being capable of releasing oneor more therapeutic agents to a wound.

2. Description of the Related Art

Dressings being capable of releasing active agent to a wound are wellknown in the art. Usually the release from these dressings is dependenton the amount of wound exudate contacting the dressing. This oftenresults in a massive release of active agent in a short period, and notin an amount being adapted to the actual need of the wound.

International patent application No. WO 03/47643 discloses a dressingfor wound treatment; the dressing comprises a therapeutic agent and abarrier layer, which separates the therapeutic agent from the woundfluid. The barrier layer comprises a substrate being degradable byspecific proteolytic enzymes in the wound fluid. Possible applicationsof this dressing are limited as the function of the dressing isdependent upon the presence of the specific enzymes.

The difficulties associated with the healing of chronic wounds may becaused by a number of factors, such as arteriosclerosis, heart disease,immune deficits, low blood supply, poor blood perfusion and sometimespoor nutrition status. Typical problems may be bacterial infections,presence of necrotic tissue and/or slough etc. One wound may suffer frommore than one of these factors, one end of the wound may have severenecrosis and the other end may suffer from infection. In order to treatsuch wound more effectively there is a need for a dressing being capableof handling these factors.

However, the therapeutic agents that are used for such wounds are oftenproteins or other sensible or fragile compounds that are not easilyincorporated in a dressing without loss of activity. Thus there is stilla need for a wound dressing being able to stabilize such agents duringprocessing and storage and providing a targeted release of the agents tothe wound.

SUMMARY OF THE INVENTION

One object of the invention is to provide a dressing being capable ofproviding a target release of one or more therapeutic agents to a wound.

Another object of the invention is to stabilize sensible therapeuticagents in wound dressings.

A third object of the present invention is to provide a wound dressingproviding an intelligent release of a therapeutical agent.

It has surprisingly been found that by incorporating liposomes intowound dressings a target drug delivery can be achieved by a triggereddelivery of therapeutic substances.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The invention relates to a wound dressing for targeted release of one ormore therapeutic ingredients, wherein the dressing comprises exudateshandling means and wherein the therapeutic ingredients are contained inliposomes, said liposomes comprising releasing means being triggered bya wound constituent and thereby releasing the therapeutic ingredients ofthe liposomes.

The wound dressing of the invention is capable of releasing one or moretherapeutic ingredients to a wound, the therapeutic ingredients beingcontained in liposomes. The wound dressing may be of any suitableconstruction, such as a foam, matrix, paste or a hydrogel, having woundexudates handling means in the form of absorbent material. The dressingmay absorb wound exudate and provide release of active ingredients fromthe liposomes. The release of active ingredient may be provided by arelease of the liposomes from the dressing, or the release may onlyconcern the agent, while the liposomes may stay in the dressing.

The dressing of the present invention may be produced in varying sizesdepending on the indication, and in an adhesive version as well as anon-adhesive version. Furthermore, the dressings may be in the form ofisland dressings, with an adhesive flange surrounding an absorbentelement, or the dressing may be in the form of a paste or gel, forcavity filling. Preferably, the dressing of the invention isconformable, soft and flexible.

The dressing may comprise a backing layer, e.g. in the form of a film.This layer may preferably be water impervious but vapor permeable. Thelayer serves as a barrier against bacteria contamination from thesurroundings, and at the same time, the vapor permeability renders itpossible for the absorbed moisture (exudates) to evaporate, and thusincrease the absorbent capacity of the dressing.

The dressing of the invention comprises wound exudates handling means,thereby providing a moist-wound healing environment.

The dressing may be suitable for any wound especially chronic woundsincluding leg ulcers, pressure sores, diabetic foot ulcers and burns.The dressing may be used on low to highly exudating wounds. Morepreferably, the dressing exhibits good retention properties so that theabsorbed wound fluid remains in the dressing even when exposed to (some)compression. In this way the surrounding skin may be protected frommaceration.

The wound exudates handling means may comprise absorbent material suchas hydrocolloids, foam, e.g. polyurethane foam, alginates, chitosan,super absorbent material, e.g. in the form of particles or fibers, fibermaterial or it may be in the form of a hydrogel. The absorbent materialmay preferably be in the form of a layer.

The absorbent layer may have an absorption capacity of 0.9% NaCl aqueoussolution at 37° C. of at least 0.05 g/cm², more preferred at least 0.1g/cm², and most preferred at least 0.2 g/cm², even most preferred atleast 0.4 g/cm². In one embodiment of the invention the absorption is atleast 0.6 g/cm².

The liposomes used in the present invention may be microscopic sphericalvesicles based on small vehicles of lipid bilayers with aqueousenvironment between the bilayers. Dependent on the constituents used andthe production method the liposomes can be made in a wide range in sizesand be either unilamellar (ULV), oligolamellar (OLV) or multilamellar(MLV).

Liposomes have been extensively investigated as drug carriers for skindelivery and parenteral delivery for many drugs. The liposomes form avery stable membrane thus providing a good protection of the activesubstance and thereby an increased flexibility of the design of thedressing may be achieved. An increased stability of the activeingredient, during production, sterilization and storage may beobtained. Furthermore, liposomes provide an opportunity to incorporateboth lipophilic and hydrophilic components without use of any additionalagents.

The liposomes may either disrupt slowly without any external mediator orcomprise releasing means being triggered by a wound constituent andthereby releasing the therapeutic ingredients of the liposomes.

Lipid components can be cholesterol or various phopholipids, lichitine.g. phosphatidyl choline, phosphatidyl ethanolamin, phosphatidyl serinand phosphatidyl inositol or mixtures thereof. As lipids may besubjected to oxidation, antioxidants may be added, e.g. tocopherol,butylhydroxitoluen, butylhydroxyianisol, ascorbic acid esters orsodiummetabisulfit.

Chronic wounds may be identified by individual indicators present in thewound, e.g. specific proteins or antigens. These may only be present inchronic non-healing wounds and are sometimes a problem in the wound andthereby the target of the therapeutic agent. Sometimes the saidindicators are present due to underlying defective condition and arethus not necessarily a problem themselves but may be an indicator of thecondition of the wound. Depending on the type of problem related to thewound, the wound constituent may be any biological constituent beingpresent in a chronic wound.

The liposomes may comprise a releasing means, said releasing means maybe triggered by a specific wound constituent and the liposomes therebyonly releasing the therapeutic agent when exposed to specific woundcomponents. The said wound constituent may only be present in chronicwounds—and may sometimes only be present in specific types of chronicwounds. The wound constituent may be in the wound exudates or bound tothe diseased cells in the wound and can be a protein or another type ofchemical or biological constituent. The wound constituent may also be acompound produced by microorganisms in the wound.

Liposomes can be custom made such that they are degraded due to thepresence of the above-mentioned indicators. The wound constituent may beable to degrade the bilipid layer of the liposomes, or it may triggermeans on the surface of the liposomes that induces transport of thetherapeutical ingredients over the membrane and thus releasing it to thewound. The wound constituent may interact with or degrade a specialcomponent incorporated in the bilayers and thus create a shuntfacilitating release of the content of the liposomes, e.g. therapeuticagents. The said agents may have several effects, e.g. a depressingeffect on the wound constituent that facilitated the release, therebycreating a self-regulated op-/down-mechanism that determines a window ofactivity of the wound component or the active agent.

The release may be induced by the action of phospholipases expressed byvarious bacteria present in the wound. Phospholipases can be dividedinto four groups depending on the position of the bond they hydrolyse onthe phospholipid substrate: phospholipases A1, A2, C and D.Phospholipases C appear to be the most important playing a significantrole in bacterial pathogenesis. Phospholipase C (originally calledlecithinase and also referred to as a-toxin) catalyzes the hydrolysis ofthe linkage between glycerol and phosphate in lecithin and otherphosphatides. The types of bacteria that commonly cause infection inwounds are Pseudomonas aeruginosa, Staphylococcus aureus and Clostridiumperfringens. All of these bacteria have been found to expressPhospholipase C. In addition several bacteria (e.g. Pseudomonasaeruginosa) has been shown to produce the phosphatidylcholine(PC)-specific phospholipase D (PLD) that catalyze the hydrolysis of PCto generate choline and phosphatidic acid. A liposome with its contentcan be prepared such that the release are dependent on degradation ofthe phospholipid bilayers by bacteria present in a wound.

In one embodiment the therapeutic ingredient may be anenzyme-controlling agent, such as a protease inhibitor. The enzyme mayonly be present in chronic wound or present in elevated levels comparedto healthy human tissue. The release of e.g. a protease inhibitordirectly into the wound may ensure a very efficient inhibition ofunwanted protease activity. The liposomes may be constructed so thatthey can diffuse to the cells deep in the wound where the inhibitors areneeded. The liposomes may exhibit an intelligent release so that theinhibitor is only released, and in the correct amount, when the proteaselevel is higher than normal. It may be specific proteases that triggerthis release. The release may then be up- and down regulated dependenton the level of the said proteases, and thereby normalizing the proteaseactivity level in a window allowing a predetermined activity. E.g. whenthe dressing is applied to a wound with (too) high protease activity,the proteases facilitate a release from the liposome. The inhibitor isreleased and will inhibit the protease in the exudate. When mostproteases are inhibited no more inhibitor are released until theprotease level may rise again.

The release may be obtained by using specific polymers in the liposomes.The released inhibitor may be a synthetic small molecule, such aspeptidomimetic or an inhibitor of biological origin, such as antibodiesand naturally occurring inhibitors. Examples of such compounds area-1-antitrypsin (AAT), anti-2-antiplasmin, alpha-2-macroglobulin,Amastatin (HCl), Aminocaproic acid, Anti-chymotrypsin, Anti-trombin,Eglin C, elafin, Epicatechin, EPI-nNE4 (DX-890), Flavonoids (catechin),GM6001, tetracycline, tissue inhibitor of metalloproteinases (TIMP's) orsecretory leukocyte protease inhibitor (SLPI).

In one embodiment the therapeutic ingredient may be a tissue healingenhancing agents or growth factor. Growth factors are essential to woundhealing because that they are involved in all of the phases of woundhealing. Growth factors specifically stimulate the migration andproliferation of cells to the wound and promote the synthesis of newtissue. Since growth factors are proteins and hence sensitive to theproduction and sterilization methods used when manufacturing dressings,it would be beneficial to incorporate these proteins in liposomes.Examples of such growth factors are platelet derived growth factor(PDGF), epidermal growth factor (EGF), transforming growth factor (TGF),insulin-like growth factor (IGF), fibroblast growth factor (FGF),keratinocyte growth factor (KGF) and vascular endothelial growth factor(VEGF) or RGD tripeptides. It may also be anabolic hormones such as,testosterone or human growth factor (hGH).

The therapeutic ingredient may comprise a pain-reliving agent such asibuprofen, ketoprofen, flurbiprofen, acetylsalicylic acid, salicylicacid, diclofenac, lornoxicam, indometacin, naproxen, paracetamol(acetaminophen), piroxicam, rofecoxib, tiaprofen acid or tolfenam acid.

In one embodiment the therapeutic ingredient may comprise abacteriostatic or bactericidal chemical compounds, e.g. iodine,iodopovidone complexes, chloramine, chlorohexidine, silver salts, zincor salts thereof. Furthermore it may comprise biologic entities such asantibacterial peptides or proteins.

The therapeutic agent may comprise an enzymatic agent. It may be anenzyme promoting wound healing by degrading undesired elements beingpresent in the wound, such as non-viable tissue, non-proliferatingcells, wound slough expressed from the immune response of the body dueto bacterial activity.

The therapeutic ingredient is a proteolytic enzyme. Proteolytic enzymesare suitable for degrading necrotic tissue and slough and therebypreparing the wound bed for a more effective healing. Examples ofsuitable proteolytic enzymes are bromelain, collagenase,deoxyribonuclease, fibrinolysin, krillase, papain, pepsin,streptodornase, streptokinase, sutilains, subtilisin, trypsin,vibralysin etc.

In one embodiment the therapeutic ingredient may be DNA or RNA. It iswell known that the delivery of DNA or RNA into cells is effectivelydone using lipid-based formulations in the form of (cationic) liposomes.This is also called gene therapy and is based on the insertion andexpression of functional genes or into a living cell, where they areexpected to produce a desired therapeutic effect. Usually the liposomesare injected to exert their effect, but by using liposomes with woundtriggered release, it could be possible to transfect cells in the woundby applying liposomes topically, released from a wound dressing. TheDNA/RNA could be genes encoding for any of the proteins mentioned above(proteases, protease inhibitors, antibacterial peptides or proteins andgrowth factors) such that the expression of the mentionedpeptides/proteins are up- or down-regulated.

An agent having a cooling effect may be comprised in the therapeuticingredient.

In one embodiment of the invention the dressing of the present inventioncomprises two or more different therapeutical agents. The differentagents may be combined in order to obtain desired properties, e.g. adressing comprising a debriding enzyme combined with a pain-killingagent. The said agents may be incorporated in liposomes with differentrelease trigger mechanisms, thereby facilitating treatment of a chronicwound where multiple disease patterns are present in either the samepart of the wound or in different local areas of the wound. Using theone and same dressing several therapeutic goals can be reached usingliposomes with different trigger-mechanisms. E.g. one part of the woundcan be treated for to high protease levels, whereas another part of thewound can be treated with growth factors etc.

EXAMPLES Example 1A Preparation of (MLV) Liposomes

Liposomes were prepared using the solvent evaporation technique. 5 ml ofan egg yolk emulsion 30% (vol/vol), obtained from Sigma-Aldrich wastransferred to a round-bottomed flask and added 20 ml of methanol. Waterand methanol was evaporated under reduced pressure at 65° C. for 15min., using the rotavapor. The lipid (approx. 1.0 ml) was diluted in 10ml of chloroform/methanol. 5 ml of this solution was dried, undernitrogen gas and 20 ml of a 5% w/vol papain PBS (phosphate bufferedsaline) solution (pH 7.4) was added. The solution was kept in a closedcontainer under nitrogen and vortexed for 30 min. thereafter it wasfrozen to −80° C. and after 5 min thawed to at 45° C. This was repeated3 times. Using flow-through dialysis with a cellulose ester dialysismembrane, with cut off at 50 kDa (Spectra/Por® MacroDialyzers fromSpectrum Laboratories, Inc, CA, USA) the untrapped papain and otherlipid components were removed. Washing was performed 3 times withPBS-buffer solution (pH 7.4). Multilamellar liposomes were obtained witha mean diameter of 2 μm, measured by light scattering measurement andthe papain content was measured to 12% (w/w) (BCA assay) and an specificactivity to 1×10⁵ USP units pr gr. (BAPA assay). The liposomes (100mg/ml) were kept in PBS buffer at 25° C. No significant change instability was observed for 3 months.

Example 1B Preparation of (ULV) Liposomes

Liposomes were prepared as in Example 1A. However, after the freeze andthaw process the solution was ultrasonificated at 60° C. at 45 min.Flow-through dialysis and washing was performed as in Example 1A.Unilamellar liposomes were obtained with a mean diameter of 100 nm, anda papain content of 2% (w/w). The liposomes (100 mg/ml) were kept in PBSbuffer at 25° C. No significant change in stability was observed for 3months.

Example 2A Foam Dressing with Liposomes Incorporated

The liposomes prepared in Example 1A and 1B were incorporated into afoam wound dressing by mixing 2 ml liposome solution, 8 g Hypol2060 (DowChemical Company), 12 g of Hypol 2002 and 18 g of water with 1% w/wPluronic 62 (BASF). The materials were mixed together for approximately15 seconds. The liquid was poured into a mould and allowed to react for10 minutes. The resulting foam sheet was dried at reduced pressure atroom temperature for 24 hours. The foam had a thickness of 3 mm and apolyurethane (PU) backing film was laminated on the top of the foam thussealing the dressing from outside. Using Franz diffusion chambers(Permgear), a release assay in saline phosphate buffer (pH 7.4) wasperformed. Protein determination (BCA assay) and activity (BAPA assay)were made. For both preparations, 95% of the enzyme was released within96 hours and no significant loss in activity was observed.

Example 2B Foam Dressing with Liposomes in a Film

1 ml of the liposome solutions prepared in Example 1A and 1B were addedto 9 ml of a PVP K90 solution (10%, w/vol) in ethanol/water (50:50,vol/vol). The solution was casted onto a release liner and the solventallowed to evaporate, thereby obtaining a film of 100 gsm (gr/m²). Therelease liner with film was applied to a foam prepared as in example 2A(but wit no liposomes). Release assay, protein content and activity wasmeasured as in example 2A. For both preparations, 95% of the enzyme wasreleased within 24 hours and no significant loss in activity wasobserved.

Example 2C Foam Dressing with Liposomes Incorporated and in a Film

A film as prepared in Example 2B was applied to a foam with liposomes asprepared in Example 2A. Release assay, protein content and activity wasmeasured as in Example 2A. For both preparations an initial burstrelease was observed, where approximately 33% of total enzyme wasrelease within 24 hours, followed by a slower release rate. After 96hours approximately 95% of the enzyme was released and no significantloss in activity was measured.

Example 2D Hydrogel Dressing with Liposomes

Liposomes were prepared as in Example 1A and 1B. The liposomes weregently suspended into a hydrogel (ratio 1:10) corresponding to EuropeanPatent No. 928 206. Using Munktell paper membranes (ooM, Grycksbo) arelease assay in Franz diffusion chambers was performed. Protein contentand activity was measured as in Example 2A. For both preparations, 95%of the enzyme was released within 36 hours and no significant loss inactivity was measured.

Example 3A Preparation of Liposomes with Release Trigged bySerum/Exudate

A hydrogel with liposomes was prepared as in Example 2D. Asandwich-ELISA assay was prepared. Release of papain from the liposomeswas measured using antibodies for papain (Goat anti-PAPAIN from ResearchDiagnostics Inc). This was done in two different release media, anordinary saline phosphate buffer (pH 7.4) and in non-deactivated calfserum (from Sigma Aldrich). The results showed that the release rate wasapproximate twice as high in calf serum than in the phosphate buffer.This indicated that constituents of the serum facilitate release fromthe liposomes. Wound exudates have to some extent similar properties andconstituents as serum and may be used as a substitute for wound exudatesin in-vitro assays, and may in this Example illustrate the impact of theexudates to the liposomes.

Example 3B Preparation of Liposomes with Targeting of Protease Activity

A hydrogel with liposomes were prepared as in Example 2D. Liposomes wereprepared as in Example 1A, however instead of papain, the inhibitor 1AAwas used and to the egg yolk emulsion was added collagen (Collagen calfskin, Type I, solution 1 mg/ml from Sigma-Aldrich). A release assay wasperformed. The release media was added protease collagenase, MMP VIII. Asandwich-Elisa assay indicated that, approximately 95% of the enzyme wasreleased within 12 hours.

Example 3C Preparation of Liposomes with Targeting of BacterialPhopholipase Activity

A hydrogel with liposomes was prepared as in Example 2D and a releaseassay was performed. The release media was added bacteria (1%Pseudomonas aeruginosa in suspension). The activity measurementindicated that for both preparations, approximately 95% of the enzymewas released within 12 hours.

1. A wound dressing for targeted release of one or more therapeuticingredients, wherein the dressing comprises exudates handling means andwherein the therapeutic ingredients are contained in liposomes, saidliposomes comprising releasing means being triggered by a woundconstituent and thereby releasing the therapeutic ingredients of theliposomes.
 2. A wound dressing according to claim 1, wherein thetherapeutic ingredient comprises a protease-controlling agent.
 3. Awound dressing according to claim 1, wherein the therapeutic ingredientcomprises a growth factor, growth hormone or other chemical/biologicalagent facilitating growth and healing of wounds.
 4. A wound dressingaccording to claim 1, wherein the therapeutic ingredient comprises anantibacterial agent.
 5. A wound dressing according to claim 1, whereinthe therapeutic ingredient comprises a pain-relieving agent.
 6. A wounddressing according to claim 1, wherein the therapeutic ingredientcomprises an enzymatic agent.
 7. A wound dressing according to claim 1,wherein the therapeutic ingredient comprises a proteolytic enzyme.
 8. Awound dressing according to claim 1, wherein the proteolytic enzymecomprises papain.
 9. A wound dressing according to claim 1, wherein thewound constituent is a biological constituent being present in a chronicwound.
 10. A wound dressing according to claim 9, wherein the woundconstituent is present in the wound exudates.